LoboVault Home

Evolution of structure and function among hotdog-fold thioesterases and HAD family phosphatases


Please use this identifier to cite or link to this item: http://hdl.handle.net/1928/17513

Evolution of structure and function among hotdog-fold thioesterases and HAD family phosphatases

Show simple item record

dc.contributor.author Min, Wang
dc.date.accessioned 2012-02-01T18:46:34Z
dc.date.available 2012-02-01T18:46:34Z
dc.date.issued 2012-02-01
dc.date.submitted December 2011
dc.identifier.uri http://hdl.handle.net/1928/17513
dc.description.abstract My doctoral research began with the function assignment to two domains of fusion protein BT4699, from Bacteriodes thetaiotaomicron, BF1314 from Bacteriodes fragilis and PG1653 from Porphyromonas gingivalis. BT4699, however, is insoluble, so its orthologue BF1314 and PG1653 were prepared and studied. The fusion proteins consist of a domain that belong to a large family of phosphatases known as Haloacid Dehalogenase (HAD) superfamily, and a domain belong to the hotdog-fold thioesterase superfamily. The genes encoding the fusion proteins located at an operon that encoding enzymes involved in the menaquinone biosynthetic pathway. So we assumed that the thioesterase domain is connected to the biosynthesis of menaquinone, which sever as a crucial role in the anaerobic bacteria respiratory chain. While the HAD domain is involved in hydrolysis of pyrophosphate produced by MenA in the lower segment of the pathway. The thioesterase domain was predicted to catalyze the hydrolysis of 1,4-dihydroxyl-2-napthoyl-CoA (DHNA-CoA) to 1,4-dihydroxyl-2-napthoate (DHNA) as the precursor of menaquinone synthesis. To study the function of thioesterase domain, the putative substrate 1,4-dihydroxy naphthoyl-CoA was prepared by chemienzymatic strategy. The structure was confirmed by H NMR and mass spectrum. HPLC analysis indicated that DHNP-CoA is very unstable, which could be self-hydrolyzed within 16 hrs at pH=7.5. By using a combination of techniques like steady-state kinetics, HPLC, and NMR, the function of thioesterase domain of the fusion protein were investigated. Steady-state kinetics studies indicated that PG1653 has high activity over DHNP-CoA. HPLC and H-NMR proved the formation of hydrolyzed products. The high specificity of BF1314 and PG1653 over DHNP-CoA testified the function of thioesterase domain. The function of HAD domain is not that as we assumed. It didn’t even show any activity towards pyrophosphate. PG1653 fusion protein did not have any phosphatase activity due to the mutation of Asp to Phe, which is a key catalytic residue of HAD phosphatase. The other fusion protein BF1314 just showed very weak activities towards a few phosphate substrates. Both truncated HAD domain and thioesterase domain were also prepared to investigate the function of HAD domain. Size exclusion chromatography was used to examine the effect of native structure on the functions of both HAD domain and thioesterase domain. The results indicated that the HAD domain may serve as an oligmerization domain that help the tetramerization of the thioesterase domain as an active thioesterase tetramer. The structure supportive role of the HAD domain disabled its phosphatase activity. The HAD domain homologue, BT3352, shares 47% identity with fusion protein BT4699. This high level of identity suggests that these two HADs are related by gene duplication. One gene copy must have fused with the thioesterase domain and took on a structural function that supports the thioesterase function while losing its catalytic function. The other copy may carry on the function of the original phosphatase. BT3352 exhibited good sugar phosphatase activities, and erythrose 4-phosphate turned out to be its physiological substrate. By comparing with its homologue yidA from E. coli, which may play a house keeping role in the galactonate degradation pathway, we hypothesized that BT3352 may also play house keeping role in likewise pathway. Bioinformatic analysis suggests that the 4 gene cluster might encode a partial β-oxidation pathway in Pseudomonas aeruginosa, which supported the pathway with seven other gene clusters. The target gene cluster contain two putative β-oxidation pathway enzymes, acyl-CoA dehydrogenase (PA5187), 3-hydroxy-acyl-CoA dehydrogenase (PA5188), a putative iron-containing alcohol dehydrogenase (PA5186) and a putative hotdog-fold thioesterase (PA5185). Substrate screen results indicated that PA5187 target C4-C15 fatty acyl-CoA, while PA5188 showed good activity towards C4-C10 and peak at 3-hydroxyhexanoyl-CoA. The hotdog-fold thioesterase exhibited narrow substrate specificities, which is good for C2-C4 acyl-CoA thioeters, and propionyl-CoA is the best substrate. Based on these results we conclude that This is a fatty acid oxidation pathway which augments the central fatty acid degradation pathway found in all bacteria. While the structure of the liganded PA5185 is needed to understand how it selects for the small substrates. en_US
dc.language.iso en_US en_US
dc.subject hotdog-fold thioesterase en_US
dc.subject Haloacid dehalogenase en_US
dc.subject menaquinone biosynthesis en_US
dc.subject beta oxidation pathway en_US
dc.subject acyl-CoA dehydrogenase en_US
dc.subject 3-hydroxyacyl-CoA dehydrogenase en_US
dc.subject.lcsh Esterases--Structure-activity relationships.
dc.subject.lcsh Haloacid dehalogenase--Structure-activity relationships.
dc.subject.lcsh Vitamin K2--Synthesis.
dc.subject.lcsh Oxidation, Physiological.
dc.title Evolution of structure and function among hotdog-fold thioesterases and HAD family phosphatases en_US
dc.type Dissertation en_US
dc.description.degree biochemistry en_US
dc.description.level Doctoral en_US
dc.description.department University of New Mexico. Dept. of Chemistry en_US
dc.description.advisor Dunaway-Mariano, Debra
dc.description.committee-member Dunaway-Mariano, Debra
dc.description.committee-member Allen, Karen N
dc.description.committee-member Mariano, Patrick S
dc.description.committee-member Wang, Wei

Files in this item

Files Size Format View
Min dissertation.pdf 3.093Mb PDF View/Open

This item appears in the following Collection(s)

Show simple item record

UNM Libraries

Search LoboVault


My Account